The ability to deliver polypeptides into various compartments of mammalian cells has obvious advantages and a wide variety of medical applications. There are several methods currently in use to deliver proteins into cells. They include the use of delivery systems (vectors) that take advantage of attenuated infectious agents that retain the ability to enter mammalian cells (viruses, bacteria), biological agents such as liposomes or dendrimers, and mechanical methods, such as needle injection of proteins or DNA directly into muscle or subcutaneous tissue.
Infectious agents, especially viruses, are highly efficient in their delivery capacity, but are limited by the size or number of proteins that can be expressed by them inside cells. In the era of AIDS and wide use of immunosuppressive chemotherapeutic agents, there is always a concern in using infectious agents to deliver products for human use, even if they are attenuated. There is also the potential for these viral vectors to induce an adverse inflammatory response in a host receiving such vectors. Liposomal systems are still not highly efficient in their cell delivery capacity, and the mechanical methods are limited by the fact that the proteins or DNA must be delivered parenterally. Delivery of foreign DNA into cells to express it inside mammalian cells offers great promise, but its limitations are many. It has to rely on high-level expression of the plasmid inside cells for the proteins to be processed properly and presented effectively, and the protein that is expressed must not undergo post-translational modification by the mammalian cell protein modification systems.
The Mycobacterium tuberculosis cell entry protein (Mce1A) protein (SEQ ID NO:2) confers on Mycobacterium tuberculosis an ability to enter mammalian cells. This protein has been described in previous patents and patent applications, including U.S. Pat. Nos. 6,214,543, 6,008,201 6,072,048, 6,224,881, and U.S. Patent Application 2001/0019716, each of which are hereby incorporated by reference in their entirety. These patents teach that by non-covalently associating the Mce1A protein and fragments thereof with heterologous pharmaceutical agents or proteins, such agents can be introduced into cells.
While useful, previous methods facilitating cell entry of heterologous proteins and particles require that the Mce1A protein or Mce1A protein fragments be purified and then attached or associated with the pharmaceutical agent. Such attachment and association reactions suffer from numerous inefficiencies including reduced activity of the pharmaceutical agent resulting from the association and/or attachment reaction.
Recent results showed that a fragment of Mce1A covalently linked as a fusion protein facilitates cell surface adhesion, and not cell entry. Inv3 (SEQ ID NO:4), a 22 amino acid fragment of the Mycobacterium cell entry protein, was covalently linked to beta-galactosidase (β-gal) and green fluorescent protein (GFP) to form Inv3 fusion proteins (Lu and Riley, Abstracts of the 98th General Meeting of the American Society for Microbiology). The Inv3-β-gal fusion proteins associated with HeLa cells. In addition, Inv3-β-gal fusion proteins that were non-covalently associated with colloid gold particles did not facilitate efficient import of the colloid gold particles into the HeLa cells, particularly in comparison to fusion proteins of InvX (SEQ ID NO:6), a 72 amino acid fragment incorporating the amino acids of Inv3.
There is thus a tremendous need to improve the efficiency of the Mce1 protein fragments as a pharmaceutical delivery agents, particularly when covalently linked to pharmaceutical delivery agents as fusion proteins capable of cell import.